Field of the invention
[0001] The present invention relates to Gemcitabine (2-deoxy-2',2'-difluorocytidine), a
known antitumor drug, and intermediates and pharmaceutically acceptable salts thereof.
Background of the invention
[0002] Gemcitabine (2-deoxy-2',2'-difluorocytidine)
is disclosed in
US 4,526,988, which discloses a synthetic method comprising the preparation of 2-deoxy-D-erythro-2,2-difluoro-ribofuranose
protected at the hydroxyls at the 3- and 5- positions with a suitable protective group
P
by reduction of 2-deoxy-D-erythro-2,2-difluoro-ribofuranos-1-ulose protected at the
hydroxyls at the 3- and 5- positions
wherein P is as defined above
with a hydride, preferably with diisobutyl-aluminium hydride in toluene (DIBAL).
[0004] US 5,945,547 indicates benzoate as a particularly preferred protective group and it claims 2-deoxy-D-erythro-2,2-difluoro-ribofuranos-1-ulose-3,5-dibenzoate,
while
US 4,965,374 claims a method for its recovery.
[0005] Protected 2-deoxy-D-erythro-2,2-difluoro-ribofuranose is subsequently transformed
into Gemcitabine by transformation of the hydroxyl at the 1 position into a leaving
group, preferably methanesulfonate, reaction with protected acetyl-cytosine to afford
protected Gemcitabine and removal of the protective groups. Gemcitabine base can then
be transformed into a pharmaceutically acceptable salt, such as the hydrochloride
usually employed in therapy.
[0006] The synthesis of high purity Gemcitabine hydrochloride, according to the regulatory
requirements contemplated by the Official Pharmacopoeias, requires purification of
the beta anomer by separation of the alpha anomer, which is difficult and often involves
costly chromatographies.
[0007] Moreover, the synthesis processes involving 2-deoxy-D-erythro-2,2-difluoro-ribofuranos-1-ulose-3,5-dibenzoate
still suffer from some problems, such as the remarkable energy waste necessary to
maintain the low temperatures (between -80 and -60°C) required during the reduction
reaction of the carbonyl group with DIBAL.
[0008] The known processes for the purification of Gemcitabine base from the hydrochloride
involve the use of high volumes of solvent (70 to 100 volumes per gram of product)
and are not ideal from the yield point of view. See in particular
US 4,965,374,
WO 2006/095359,
WO 2005/095430 and
WO 2007/049294.
Disclosure of the invention
[0009] In a first aspect, the invention relates to a process for the preparation of 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate:
by reduction of 2-deoxy-D-erythro-2,2-difluoro-ribofuranos-1-ulose-3,5-dibenzoate
with calcium or sodium borohydride.
[0010] This reaction can be carried out with methods and reagents known to those skilled
in the art; in particular aprotic polar solvents, e.g. tetrahydrofuran, ethyl acetate
and dioxane, at a temperature ranging from -20 to +20°C, can be used; after completion
of the reaction, 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate can be
transformed into Gemcitabine according to known methods, for example according to
the process disclosed in
US 4,965,347, which comprises the transformation of the hydroxyl at the 1- position into a methanesulfonate
group, the reaction with bis-trimethylsilyl-N-acetyl-cytosine in the presence of trimethylsilyl
triflate and the removal of the protective groups. Examples 4 - 8 reported in the
Experimental Section of the present application disclose in further detail the transformation
of the intermediate into Gemcitabine according to the teaching of said Patent.
[0011] The preparation of 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate by
reduction of the precursor with calcium or sodium borohydride is particularly convenient
from the industrial point of view in that it reduces the energy requirements of the
process, as extremely low temperatures are no longer necessary.
[0012] In a second aspect, the invention relates to a process for the preparation of Gemcitabine
hydrochloride in conformity with the pharmacopoeia requirements. The expression "in
conformity with the pharmacopoeia requirements" means Gemcitabine hydrochloride having
purity not lower than 99.8%, with Gemcitabine α anomer <0.1%, cytosine <0.1%, any
other impurity <0.1%, total impurities <0.2% and titer ranging from 97.5% to 101.5.
Said process comprises the purification of crude Gemcitabine (obtained according to
known methods or using 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate
prepared as described above), on an ion exchange chromatographic column, the dissolution
of the eluted Gemcitabine in methanol and the precipitation of the hydrochloride by
addition of concentrated HCl (37%) and ethyl acetate. Chromatography is carried out
with commercially available reagents and according to procedures known to those skilled
in the art. The resin usually employed is a strong cation exchange resin; according
to a particular embodiment, the resin is Amberlite IR 120. Typically, the resin is
conditioned with water, after that a crude Gemcitabine aqueous solution is dissolved
in water, loaded on the resin and eluted with an ammonia aqueous solution at increasing
concentrations ranging from 5 to 20% by weight. The eluate is evaporated to dryness
and the oil which is usually obtained, consisting of Gemcitabine base, is dissolved
in methanol; then added with a 37% hydrochloric acid aqueous solution in stoichiometric
amounts and an ethyl acetate volume approximately equivalent to the volume of the
methanol solution; rapid formation of a precipitate, consisting of the hydrochloride,
is observed. Usually, the mixture is left to stand overnight at a temperature of 5°C
to improve yields, before filtering and drying the product.
[0013] In a third aspect, the invention relates to a process for the preparation of high
purity Gemcitabine hydrochloride starting from crude Gemcitabine (obtained according
to known methods or using 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate
prepared as described above). This process comprises the dissolution of crude Gemcitabine
in methanol and the subsequent addition of a concentrated hydrochloric acid aqueous
solution and acetone or ethyl acetate; after addition of concentrated hydrochloric
acid rapid formation of a precipitate is observed; then a volume of acetone or ethyl
acetate equal to the methanol volume is added. Rapid formation of a precipitate, consisting
of the hydrochloride, is observed. Usually, the mixture is left to stand overnight
at a temperature of 5°C before filtering and drying the product. Il volume of the
crystallization solvent mixture is preferably of approx. 30 volumes per gram of product.
The resulting hydrochloride usually as HPLC purity higher than 95% and lower than
98%.
[0014] In a fourth aspect, the invention relates to a process for the preparation of Gemcitabine
hydrochloride in conformity to the Pharmacopoeia requirements, comprising the recrystallization
of Gemcitabine hydrochloride from water, methanol and acetone or from water, methanol
and ethyl acetate.
[0015] The volume of the crystallization solvent mixture is preferably of approx. 30 volumes
per gram of product.
[0016] This process can be applied either starting from Gemcitabine hydrochloride with low
HPLC titer, for example with titer below 85%, as illustrated in Example 12 of the
present application, or starting from Gemcitabine hydrochloride with higher purity,
obtained as described in the preceding paragraph. Comparison between the purity values
and HPLC titer and content in the alpha form reported in Examples 11 and 12 and the
values reported in Example 13 evidences that the use of water/methanol/acetone or
water/methanol/ethyl acetate ternary mixtures for the recovery and recrystallization
of Gemcitabine hydrochloride is remarkably advantageous over the use of a binary mixture
consisting of water and acetone. The water/methanol/ethyl acetate mixture is particularly
preferred.
[0017] In a fifth aspect, the invention relates to a process for the preparation of Gemcitabine
hydrochloride comprising the following steps:
- a) reduction of 2-deoxy-D-erythro-2,2-difluoro-ribofuranos-1-ulose-3,5-dibenzoate
with calcium or sodium borohydride, to afford 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate:
- b) transformation of the hydroxyl at the 1- position into a leaving group and reaction
with acetylcytosine to afford Gemcitabine base;
- c) purification of Gemcitabine base by ion exchange chromatography;
- d) dissolution of Gemcitabine base obtained at step c) in methanol and subsequent
addition of a concentrated hydrochloric acid aqueous solution and acetone or ethyl
acetate to afford a precipitate consisting of Gemcitabine hydrochloride;
- e) recovery of Gemcitabine hydrochloride obtained at step d) and recrystallization
from water, methanol and acetone or from water, methanol and ethyl acetate.
[0018] In step e) the use of a water, methanol and ethyl acetate mixture, in the ratios
specified above, is preferred.
[0019] This process is particularly suitable from the industrial point of view in that it
combines the advantages of the synthesis of the intermediate and those of the recovery
and purification of the hydrochloride according to the invention.
[0020] The process of the invention is more advantageous than those disclosed in
US 4,965,374,
WO 2006/095359,
WO 2005/095430 and
WO 2007/049294 from the standpoint of solvent volumes and tolerability thereof, which is markedly
higher than that of monoglyme and dioxane described in
WO 2007/049294 and of acetonitrile described in
WO 2005/095430 and
WO 2006/095359. Furthermore, the process of the invention has better efficiency and selectivity
over the known processes as the recovery of the crude provides a product which is
already 95-97% pure and which attains the desired quality through a single subsequent
operation. Conversely, in
WO 2005/095430 and
WO 2006/095359 the crude has to be slurried in water before crystallization.
US 4,965,374 provides a product with satisfactory quality only after repeated, complex operations,
as reported by way of comparison in the Examples.
[0021] The experimental section shows in detail the invention.
EXPERIMENTAL SECTION
Example 1 - Preparation of 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate
with Ca(BH4)2
[0022] 0.19 g of CaCl
2 (1.99 mmoles) and 0.151 g of NaBH
4 (3.99 mmoles) were added to 4 ml of EtOH. The suspension was cooled to -20°C and
added with a solution of 2-deoxy-D-erythro-2,2-difluoro-pentafuranos-1-ulose-3,5-dibenzoate
(5.0 g; 13.29 mmoles) in 20 ml of tetrahydrofuran during 45 minutes. After a further
60 minutes, the reaction was monitored by TLC (1:1 ethyl acetate: hexane) and after
completion it was arrested by addition of 20 ml of ethyl acetate followed by concentrated
HCl to pH=2. After stirring for 10 minutes at room temperature, the aqueous phase
was separated and repeatedly extracted with ethyl acetate (20 ml), afterwards the
combined organic phases were washed with brine (20 ml) and 5% NaHCO
3 aqueous solution (20 ml). The organic phase was concentrated under vacuum to give
a thick oil (5.5 g). This product contained an impurity less polar than the product.
Example 2 - Preparation of 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate
with NaBH4
[0023] 100 g of 2-deoxy-D-erythro-2,2-difluoro-pentafuranos-1-ulose-3,5-dibenzoate (0.266
mole) was dissolved in a 2 L round-bottom flask in 650 ml of tetrahydrofuran and cooled
to 2°C, after which 3.20 g of NaBH
4 (0.084 mole) was added. The suspension was stirred for 3h, then a sample was taken
and monitored by HPLC. After completion, the reaction was stopped by addition of 500
ml of brine and concentrated HCl to pH 2 (about 7 ml). The mixture was stirred for
10 minutes, then the aqueous phase was separated at room temperature; the organic
phase was washed with 500 ml of brine, added with a further 500 ml of brine and the
biphasic system was evaporated to remove THF; when an oil suspended in water was obtained,
500 ml of CH
2Cl
2 were added thereto. The mixture was stirred for 15 minutes, then the aqueous phase
was separated and extracted again with 150 ml of CH
2Cl
2, then the combined organic phases were concentrated to give an oil (107 g). HPLC
analysis showed purity of 75%; the calculated yield was 90%. Part of the oil was subjected
to silica gel chromatography (eluent: 6:4 ethyl acetate-hexane) to give 3.0 g of pure
lactol and 300 mg of impurity.
Example 3 - Preparation of 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate
with NaBH4
[0024] 10 g of 2-deoxy-D-erythro-2,2-difluoro-pentafuranos-1-ulose-3,5-dibenzoate (26.6
moles) was dissolved in a 250 ml round-bottom flask in 70 ml of AcOEt and cooled a
2°C, then added with 0.32 g of NaBH
4 (8.4 moles). The resulting suspension was stirred for two hours, then a sample was
taken and monitored by HPLC. After completion, the reaction was stopped by addition
of 70 ml of brine and concentrated HCl to pH 2 (about 7 ml). The mixture was stirred
for 10 minutes, then the aqueous phase was separated at room temperature. The organic
phase was washed with 70 ml of brine, then added with a further 70 ml of brine, separated
from the aqueous phase and concentrated to give an oil (10g). HPLC analysis showed
purity of 75%. The calculated yield was 88%.
Example 4 - Preparation of 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate-1-methanesulfonate
[0025] 101 g of 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate in the form of
an oil (0.227 moles calculated) was dissolved in a 2 L round-bottom flask in 600 ml
of methylene chloride. This mixture was cooled at 0 - +5°C and kept under stirring,
then added with 42.8 g of triethylamine (0.424 mole) and 37.4 g of methanesulfonyl
chloride (0.327 mole) during 30 minutes. The reaction was stirred a 5°C for 1h, then
a sample was taken and monitored by HPLC. After completion, the reaction was added
with 250 ml of water and 7 ml of concentrated HCl to pH 2. The mixture was stirred
for 10 minutes, then the phases were separated and the organic one was washed twice
with 250 ml of brine (conc. 10%). The solution was concentrated to give an oil (115
g). HPLC analysis showed purity of 75%. The calculated yield was 80% on the lactone.
Example 5 - Preparation of 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate-1-methanesulfonate
[0026] 5.60 g 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate in the form of
an oil obtained according to Example 3 (11.96 mmoles calculated) was dissolved in
250 ml a round-bottom flask in 60 ml of ethyl acetate. The mixture was cooled to 0
- +5°C, added with 2.68 g of triethylamine (26.58 mmoles) and 1.52 g of methanesulfonyl
chloride (13.,29 moles) during 30 minutes. The reaction was stirred a 5°C for 1h,
then a sample was monitored by means of TLC (toluene/MeOH 9:1). After completion,
50 ml of water and 20 ml of AcOEt were added and the mixture was stirred for 10 minutes,
then the phases were separated and the organic one was added with 2.6 ml of 18% HCl
to pH 1. The mixture was stirred for 10 minutes, then the phases were separated and
the organic one was washed with 50 ml of brine, then concentrated to give 6.2 g of
a residual oil.
Example 6 - Preparation of 2'-deoxy-2',2'-difluoro-N-1-acetyl cytidine-3',5'-dibenzoate
[0027] Following the process disclosed in
US 4,965,374 and using the following reagents: 88.7 g of acetyl cytosine (0.579 mole), 280 g of
hexamethyldisilazane (1.735 moles), 4.4 g of ammonium sulfate (0.033 mole), 116 g
of trimethylsilyl triflate (0.522 mole), 115 g of mesylate obtained according to Example
4 (0.202 mole calculated), 135 g of a thick oil were obtained, which contained 52.1
g of protected Gemcitabine (determined by HPLC).
Example 7 - Preparation of 2'-deoxy-2',2'-difluoro-N-1-acetyl cytidine-3',5'-dibenzoate
[0028] Following the process disclosed in
US 4,965,374 and using the following reagents: 83.2 g of acetyl cytosine (0.543 mole), 333 g of
hexamethyldisilazane (2.063 moles), 3.3 g of ammonium sulfate (0.025 mole), 132 g
of trimethylsilyl triflate (0.594 mole), 115 g of mesylate (obtained according to
Example 4) (0.202 mole calculated), 118 g of a thick oil were obtained.
Example 8 - Preparation of crude Gemcitabine base
[0029] Following the procedure disclosed in
US 4,965,374, with 45.5 g of oil obtained according to Example 6, 15 g of a crude oil was obtained;
HPLC analysis showed titer of 26.06% in the form β (3.78 g) and an α/β ratio = 1.29.
Example 9 - Purification of Gemcitabine on ion exchange resin
[0030] 80 ml of an aqueous solution containing 12.5 g of crude Gemcitabine free base was
eluted over 230 ml of resin IR120 (capacity 1.9 meq/ml) and the resin was washed with
700 ml of water; the eluate contained impurities. Gemcitabine was eluted with 400
ml of 7.5% (w/w) ammonia solution in water, then elution was continued using 200 ml
of a 13% ammonia solution and subsequently 100 ml of a 15% solution. The fractions
containing the product were concentrated and dissolved in 60 ml of MeOH. This solution
was added with 6 ml of conc. HCl and 70 ml of AcOEt and a precipitate quickly formed.
The mixture was stirred for 1h at room temperature and overnight at 5°C. The solid
(Gemcitabine HCl) was filtered, washed with MeOH and dried under vacuum at 45°C overnight
(yield: 4.20 g; HPLC purity = 98.8%; form α 3.3%; HPLC purity = 96.35%.
Example 10 - Preparation of Gemcitabine HCl
[0031] The oil obtained according to Example 8 (15 g) was added with 45 ml of MeOH and 3.7
g of 37% HCl at room temperature, after a few minutes a precipitate formed. After
addition of 45 ml of AcOEt, the mixture was stirred for 2h at room temperature, then
overnight at 5°C. The solid was filtered and washed with 1 ml of cold MeOH and 2x2
ml of AcOEt to give 2.79 g of form β (HPLC purity: 97%; area corresponding to the
content in form α: 2.7%).
Example 11 - Preparation of Gemcitabine HCl
[0032] 15 g of crude Gemcitabine free base obtained according to Example 8 was added with
45 ml of MeOH and 3.7 g of 37% HCl; after a few minutes a precipitate formed. After
addition of 45 ml of acetone, the mixture was stirred for two hours 2h at room temperature,
then overnight at 5°C. The solid was filtered and washed with 1 ml of methanol MeOH
and twice with 2 ml of acetone, to give 2.58 g of product enriched in the form β (HPLC
purity 97%; area corresponding to the content in form α: 3%).
Example 12 - Purification of Gemcitabine HCl from a water-MeOH-AcOEt ternary mixture
[0033] 3.5 g of Gemcitabine HCl (titer 83%; form β 95%, form α 5%) was dissolved in 10.5
ml of water and 30 ml of MeOH al 78-79°C; the resulting clear solution was cooled
to 70°C, then added with 42 ml of AcOEt. The mixture was cooled at room temperature
and stirred for 2h, then cooled at 5°C for 1h. The solid was filtered, washed with
AcOEt and dried under vacuum at 45°C, to give 2.74 g of product (HPLC purity 99.9%;
form α lower than 0.1%. HPLC titer = 102.4%.).
Example 13 - Purification of Gemcitabine HCl from a water/acetone mixture (reference
example according to US 4,965,374)
[0034] 3.5 g of Gemcitabine HCl (titer = 83%; form β 95%, form α 5%) was dissolved in 14
ml of water at 90°C; the clear solution was cooled to 50°C, then added with 350 ml
of acetone. The mixture was cooled at room temperature, stirred for 2h, cooled at
5°C overnight and the product was filtered and dried at 45°C overnight (yield: 3.14
g; HPLC purity = 99.27%; form α = 0.54%. HPLC titer = 96.20%).
1. Process for the preparation of 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate
comprising the reduction of 2-deoxy-D-erythro-2,2-difluoro-ribofuranos-1-ulose-3,5-dibenzoate
with sodium or calcium borohydride.
2. The process according to claim 1 wherein the reaction is carried out in a polar aprotic
solvent and at a temperature ranging from -20 to +20°C.
3. The process according to claim 2 in which in solvent is selected from tetrahydrofuran,
ethyl acetate and dioxane.
4. Process for the preparation of Gemcitabine hydrochloride
which comprises the purification of crude Gemcitabine by ion exchange chromatography,
the dissolution of the obtained Gemcitabine in methanol and the precipitation of Gemcitabine
hydrochloride from concentrated hydrochloric acid and ethyl acetate.
5. The process as claimed in claim 4 wherein the resin is a strong cation exchange resin.
6. The process as claimed in claim 5 which is carried out using a stoichiometric amount
of concentrated hydrochloric acid and the same volume of ethyl acetate as the methanol
volume necessary for dissolving the Gemcitabine obtained from the elution.
7. Process for the purification of Gemcitabine hydrochloride comprising the recrystallization
of Gemcitabine hydrochloride from water, methanol and acetone or from water, methanol
and ethyl acetate.
8. Process for the preparation of Gemcitabine hydrochloride comprising the following
steps:
a) the reduction of 2-deoxy-D-erythro-2,2-difluoro-ribofuranos-1-ulose-3,5-dibenzoate
with calcium or sodium borohydride to afford 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate:
b) the transformation of the hydroxyl group at the 1- position into a leaving group
and the reaction with acetylcytosine to give Gemcitabine base;
c) the purification of Gemcitabine base by ion exchange chromatography;
d) the dissolution of Gemcitabine base obtained at step c) in methanol and the subsequent
addition of an aqueous solution of concentrated hydrochloric acid and acetone or ethyl
acetate to give a precipitate consisting of Gemcitabine hydrochloride;
e) the recovery of Gemcitabine hydrochloride obtained at step d) and the recrystallization
from water, methanol and acetone or water, methanol and ethyl acetate.